New Sequencing technologies help revealing unexpected mutations in Autosomal Dominant Hypercholesterolemia

Sandy Elbitar,Gina Peloso,Petra El Khoury,Catherine Boileau,Ali Ben-Djoudi-Ouadda,Josée Hamelin,Nabil G Seidah,Mathilde Varret,Delia Susan-Resiga,Youmna Ghaleb,Jean-Pierre Rabès,Valérie Carreau,Eric Bruckert,Marianne Abifadel,Nathan Stitziel

doi:10.1038/s41598-018-20281-9

Abstract

Autosomal dominant hypercholesterolemia (ADH) is characterized by elevated LDL-C levels leading to coronary heart disease. Four genes are implicated in ADH: LDLR, APOB, PCSK9 and APOE. Our aim was to identify new mutations in known genes, or in new genes implicated in ADH. Thirteen French families with ADH were recruited and studied by exome sequencing after exclusion, in their probands, of mutations in the LDLR, PCSK9 and APOE genes and fragments of exons 26 and 29 of APOB gene. We identified in one family a p.Arg50Gln mutation in the APOB gene, which occurs in a region not usually associated with ADH. Segregation and in-silico analysis suggested that this mutation is disease causing in the family. We identified in another family with the p.Ala3396Thr mutation of APOB, one patient with a severe phenotype carrying also a mutation in PCSK9: p.Arg96Cys. This is the first compound heterozygote reported with a mutation in APOB and PCSK9. Functional studies proved that the p.Arg96Cys mutation leads to increased LDL receptor degradation. This work shows that Next-Generation Sequencing (exome, genome or targeted sequencing) are powerful tools to find new mutations and identify compound heterozygotes, which will lead to better diagnosis and treatment of ADH.

Highlights

Hypercholesterolemia is a major risk factor for atherosclerosis and its premature cardiovascular complications
A study in a French cohort, which included probands and families with hypercholesterolemia recruited through the French Research Network for Autosomal dominant hypercholesterolemia (ADH) from several regions of France, showed that the LDLR gene is implicated in 73.9% of the cases, while mutations in APOB and PCSK9 are responsible of 6.6% and 0.7% of the cases respectively
We show that new sequencing technologies like exome sequencing are powerful tools to find new mutations in genes already known to be implicated in ADH, APOB in particular, especially because the Sanger sequencing strategies classically performed in ADH genetic diagnosis do not target the entire region of APOB

Summary

Introduction

Hypercholesterolemia is a major risk factor for atherosclerosis and its premature cardiovascular complications. Autosomal dominant hypercholesterolemia (ADH) (MIM # 143890) is one of the most common monogenic disorders It is caused by mutations in genes encoding key proteins involved in the LDL receptor (LDLR) endocytic and recycling pathways, causing decreased cellular uptake of LDL and increased plasma LDL-cholesterol (LDL-C) concentrations. A study in a French cohort, which included probands and families with hypercholesterolemia recruited through the French Research Network for ADH from several regions of France, showed that the LDLR gene is implicated in 73.9% of the cases, while mutations in APOB and PCSK9 are responsible of 6.6% and 0.7% of the cases respectively. Concerning the APOB gene, the p.Arg3527Gln, known as APOB3527 or APOB3500, is the first and most common ADH-related mutation in APOB reported in late 1980s3 It is responsible alone for more than 95% of Familial Defective apolipoprotein B cases (FDB)[14] (MIM# 144010). This gene was not classically entirely studied in ADH by Sanger sequencing and routinely only a fragment of exon 26 and another of exon 29 are analyzed, covering the regions where the functional mutations causing hypercholesterolemia have been described[15,16]

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